WO1996024343A1 - Utilisation de composes specifiques comportant lck sh2 pour le traitement de maladies auto-immunes et du rejet d'allogreffes - Google Patents

Utilisation de composes specifiques comportant lck sh2 pour le traitement de maladies auto-immunes et du rejet d'allogreffes Download PDF

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WO1996024343A1
WO1996024343A1 PCT/US1996/001964 US9601964W WO9624343A1 WO 1996024343 A1 WO1996024343 A1 WO 1996024343A1 US 9601964 W US9601964 W US 9601964W WO 9624343 A1 WO9624343 A1 WO 9624343A1
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domain
binding affinity
human
binds
lck
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PCT/US1996/001964
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Damien John Dunnington
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Smithkline Beecham Corporation
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Priority to EP96905494A priority Critical patent/EP0809490A4/fr
Priority to BR9607614A priority patent/BR9607614A/pt
Priority to AU49237/96A priority patent/AU4923796A/en
Priority to JP8524486A priority patent/JPH10513474A/ja
Publication of WO1996024343A1 publication Critical patent/WO1996024343A1/fr
Priority to FI973259A priority patent/FI973259A/fi
Priority to NO973659A priority patent/NO973659L/no
Priority to MXPA/A/1997/006130A priority patent/MXPA97006130A/xx

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the src-related, lymphocyte-specific protein tyrosine kinase, p56" k (lck) is physically associated through its unique amino-terminal domain with the cytoplasmic tail of CD4 in T lymphocytes.
  • the CD4 associated lck is an essential component in the T cell activation cascade, and an intact lck SH2 domain/CD4 complex is required for functional immune response of T cells to antigen. Inhibition of the lck SH2 domain/CD4 complex results in T cell-specific immunosuppression.
  • a number of polypeptide growth factors and hormones mediate their cellular effects through a signal transduction pathway.
  • Tyrosine phosphorylation may be the primary, or possibly even the sole, indicator of signal transduction in multicellular organisms.
  • Receptor-bound and intracellular PTKs regulate cell proliferation, cell differentiation and signalling processes in immune system cells. Aberrant protein tyrosine kinase activity has been implicated or is suspected in a number of pathologies such as diabetes, atherosclerosis, psoriases, septic shock, bone loss, anemia, many cancers and other proliferative diseases.
  • tyrosine kinases and the signal transduction pathways which they are part of are potential targets for drug design.
  • Many of the proteins comprising signal transduction pathways are present at low levels and often have opposing activities.
  • the properties of these signalling molecules allow the cell to control transduction by means of the subcellular location and juxtaposition of effectors as well as by balancing activation with repression such that a small change in one pathway can achieve a switching effect.
  • the formation of transducing complexes by juxtaposition of the signalling molecules through protein-protein interactions are mediated by specific docking domain sequence motifs.
  • SH2 domains Src homology 2 domains, which are conserved non- catalytic sequences of approximately 100 amino acids found in a variety of signalling molecules such as non-receptor PTKs and kinase target effector molecules and in oncogenic proteins, play a critical role.
  • the SH2 domains are highly specific for short phosphotyrosine-containing peptide sequences found in autophosphorylated PTK receptors or intracellular tyrosine kinases.
  • Antagonism of the src SH2 domain is indicated as effecting bone resorption while antagonism of the lck SH2 domain (discussed herein) or the fyn SH2 domain induces immunosuppression.
  • the inhibition of bone resorption would be undesirable in long term therapy which requires the induction of immunosuppression.
  • selective lck SH2 domain antagonists can be identified by binding assays against the subset of SH2 domains consisting of; the src SH2 domain, the lck SH2 domain, the fyn SH2 domain, the SHPTP2 SH2 domain, the p85 domain, the Grb2 SH2 domain and the hep SH2 domain.
  • the present invention provides a method of treating autoimmune diseases in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human lck SH2 domain with a binding affinity greater than fifty-fold higher than the binding affinity with which the compound binds to a human src SH2 domain and a human fyn SH2 domain, and (b) binds to a human hep SH2 domain, a human Grb2 domain, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than fifty-fold lower than the binding affinity with which the compound binds to such lck SH2 domain.
  • the present invention also provides a method of inhibiting allograft rejection in a subject which comprises administering to the subject an allograft rejection inhibiting amount of a compound which (a) binds to a human lck SH2 domain with a binding affinity greater than fifty-fold higher than the binding affinity with which the compound binds to a human src SH2 domain and a human fyn SH2 domain, and (b) binds to a human hep SH2 domain, a human Grb2 domain, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than fifty-fold lower than the binding affinity with which the compound binds to such lck SH2 domain.
  • the present invention also provides a method of inducing immunosuppression in a subject which comprises administering to the subject an immunosuppression inducing amount of a compound which (a) binds to a human lck SH2 domain with a binding affinity greater than fifty-fold higher than the binding affinity with which the compound binds to a human src SH2 domain and a human fyn SH2 domain, and (b) binds to a human hep SH2 domain, a human Grb2 domain, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than fifty-fold lower than the binding affinity with which the compound binds to such lck SH2 domain.
  • autoimmune diseases means any disorder characterized by an overly active immune response or an immune response wrongly directed against self antigen.
  • the preferred autoimmune disease is rheumatoid arthritis.
  • Other diseases such as multiple sclerosis, systemic lupus erythematosis and type I diabetes are also included.
  • treating means prophylactic or therapeutic therapy.
  • compound means a nonpeptide chemical compound.
  • the term "lck SH2 domain antagonists” means a compound which (a) binds to a human lck SH2 domain with a binding affinity greater than fifty-fold higher, preferably greater than one hundred ⁇ fold higher, than the binding affinity with which the compound binds to a human src SH2 domain and a human fyn SH2 domain, and (b) binds to a human hep SH2 domain, a human Grb2 SH2, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than fifty-fold lower, preferably greater than one hundred-fold lower, than the binding affinity with which the compound binds to such lck SH2 domain.
  • the present invention provides a method of treating autoimmune diseases in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human lck SH2 domain with a binding affinity greater than fifty-fold higher than the binding affinity with which the compound binds to a human src SH2 domain and a human fyn SH2 domain, and (b) binds to a human hep SH2 domain, a human Grb2 SH2, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than fifty-fold lower than the binding affinity with which the compound binds to such lck SH2 domain.
  • a preferred aspect of the invention provides a method of treating autoimmune diseases in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human lck SH2 domain with a binding affinity greater than fifty-fold higher than the binding affinity with which the compound binds to a human src SH2 domain and a human fyn SH2 domain.
  • a preferred aspect of the invention provides a method of treating autoimmune diseases in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human lck SH2 domain with a binding affinity greater than one hundred-fold higher than the binding affinity with which the compound binds to a human src SH2 domain and a human fyn SH2 domain, and (b) binds to a human hep SH2 domain, a human Grb2 SH2, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than one hundred-fold lower than the binding affinity with which the compound binds to such lck SH2 domain.
  • a preferred aspect of the invention provides a method of treating autoimmune diseases in a subject which comprises administering to the subject a therapeutically effective amount of a compound which (a) binds to a human lck SH2 domain with a binding affinity greater than one hundred-fold higher than the binding affinity with which the compound binds to a human src SH2 domain and a human fyn SH2 domain.
  • the invention also provides a method of inhibiting allograft rejection in a subject which comprises administering to the subject an allograft rejection inhibiting amount of a compound which (a) binds to a human lck SH2 domain with a binding affinity greater than fifty-fold higher, preferably greater than one hundred-fold higher, than the binding affinity with which the compound binds to a human src SH2 domain and a human fyn SH2 domain, and (b) binds to a human hep SH2 domain, a human Grb2 domain, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than fifty-fold lower, preferably greater than one hundred-fold lower, than the binding affinity with which the compound binds to such lck SH2 domain.
  • a preferred aspect of the invention provides a method of inhibiting allograft rejection in a subject which comprises administering to the subject an allograft rejection inhibiting amount of a compound which (a) binds to a human lck SH2 domain with a binding affinity greater than fifty-fold higher, preferably greater than one hundred-fold higher, than the binding affinity with which the compound binds to a human src SH2 domain and a human fyn SH2 domain.
  • the invention also provides a method of inducing immunosuppression in a subject which comprises administering to the subject an immunosuppression inducing amount of a compound which (a) binds to a human lck SH2 domain with a binding affinity greater than fifty-fold higher, preferably greater than one hundred ⁇ fold higher, than the binding affinity with which the compound binds to a human src SH2 domain and a human fyn SH2 domain, and (b) binds to a human hep SH2 domain, a human Grb2 domain, a human SH-PTP2 SH2 domain and a human p85 SH2 domain with a binding affinity which is greater than fifty-fold lower, preferably greater than one hundred-fold lower, than the binding affinity with which the compound binds to such lck SH2 domain.
  • a preferred aspect of the invention provides a method of inducing immunosuppression in a subject which comprises administering to the subject an immunosuppression inducing amount of a compound which (a) binds to a human lck SH2 domain with a binding affinity greater than fifty-fold higher, preferably greater than one hundred-fold higher, than the binding affinity with which the compound binds to a human src SH2 domain and a human fyn SH2 domain.
  • the inhibitory activity of compounds at the different human SH2 domains was determined in vitro using SH2 domains expressed as fusion proteins in __ coli as further described in detail in Example 11 below.
  • the IL2 Production Assay which measures the production of IL2 in T cells in response to various stimuli.
  • the most commonly used T cell is the Jurkat human T cell line.
  • the Jurkat is stimulated with, a) mitogenic lectin (phytohemaglutinin or PHA) plus calcium inonophore or plus phorbol esters, or b) T cell receptor antibody plus phorbol esters.
  • the measurement of IL2 by ELISA specific for human IL2 indicates the level of activity and 2)
  • MLR Three way Human Mixed Lymphocytes Reaction
  • This assay measures the response of lymphocytes from one individual to the foreign antigens present in blood cells of another individual.
  • the lymphocytes from the blood of three different donors (3- way response) are mixed together and incubated in complete growth medium for 4 - 5 days.
  • the determination cell proliferative response by measuring thymidine incorporation indicates
  • Activity in these assays is recognized in the art as correlating with efficacy in treating autoimmune diseases in vivo. Activity in these assays is also recognized in the art as correlating with efficacy in inhibiting allograft rejection in vivo. Activity in these assays is also recognized in the art as correlating with efficacy in inducing immunosuppression in vivo.
  • the present invention therefore provides a method of inducing immunosuppression, which comprises administering a quantity of a lck SH2 domain antagonists defined as herein in a quantity effective to induce immunosuppression.
  • the drug may be administered to a patient in need of immunosuppression by any conventional route of administration, including, but not limited to, intravenous, intramuscular, oral, subcutaneous, intradermal, and parenteral.
  • the quantity effective to induce immunosuppression is from about 0.001 mg per kg to about 10.0 mg per kg of subject body weight.
  • the selected dose will be an efficacious, nontoxic quantity selected from about 0.001 mg per kg to about 10.0 mg per kg of subject body weight.
  • the selected dose will be administered from about 1-6 times daily.
  • compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixers, and suspensions.
  • forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
  • the present invention further provides a method of inhibiting allograft rejection, which comprises administering a quantity of a lck SH2 domain antagonists defined as herein in a quantity effective to inhibit allograft rejection.
  • the drug may be administered to a patient with a recently implanted allograft or in anticipation of receiving an allograft by any conventional route of administration, including, but not limited to, intravenous, intramuscular, oral, subcutaneous, intradermal, and parenteral.
  • the quantity effective to inhibit allograft rejection is from about 0.001 mg per kg to about 10.0 mg per kg of subject body weight.
  • the selected dose will be an efficacious, nontoxic quantity selected from about 0.001 mg per kg to about 10.0 mg per kg of subject body weight.
  • the selected dose will be administered from about 1-6 times daily.
  • compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixers, and suspensions.
  • forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
  • the drug may otherwise be prepared as a sterile solid composition which may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.
  • Carriers are intended to include necessary and inert binders, suspending agents, lubricants, flavorants, sweeteners, preservatives, dyes and coatings.
  • Optimal dosages to be administered may be readily determined by those skilled in the art, and will vary with the particular lck SH2 domain antagonist in use, the strength of the preparation, the mode of administration, and the advancement of the disease condition. Additional factors depending on the particular patient being treated will result in a need to adjust dosages, including patient age, weight, diet, and time of administration.
  • the invention also provides for the use of a lck SH2 domain antagonists in the manufacture of a medicament for use in the treatment of autoimmune diseases.
  • the invention also provides for the use of a lck SH2 domain antagonists in the manufacture of a medicament for use in inhibiting allograft rejection.
  • the invention also provides for the use of a lck SH2 domain antagonists in the manufacture of a medicament for use in inducing immunosuppression.
  • the invention also provides for a pharmaceutical composition for use in the treatment of autoimmune diseases which comprises a lck SH2 domain antagonists.
  • the invention also provides for a pharmaceutical composition for use in inhibiting allograft rejection which comprises a lck SH2 domain antagonists.
  • the invention also provides for a pharmaceutical composition for use in inducing immunosuppression which comprises a lck SH2 domain antagonists.
  • L-3,5-Dibromotyrosine can be prepared by methods known in the art, for example as described in "Thyoid Hormones and Analogues. I. Synthesis, Physical Properties and Theoretical Calculations” E. C. Jorgensen, Hormonal Proteins and Peptides, Vol. VI, 1978, Academic Press, N.Y. and references cited therein. L-3,5-dibromo-N-trifluoroacetyl-tyrosine methyl ester (for use in Example 2
  • Example 2B (b)) can be prepared according to the following procedure.
  • L-3,5-Dibromotyrosine 500 g was suspended in methanol (5 liters) and dry hydrogen chloride passed through the stirred suspension for 5 hours. The reaction mixture was evaporated to dryness, the residue suspended in water (4 liters), and the pH adjusted to 6 with 40% sodium hydroxide. The precipitate was collected and washed with water to give L-3,5-dibromotyrosine methyl ester (467 g, 90%), m.p. 201°-203°. The ester (768 g) was suspended in chloroform (2.7 liters) and ethyl acetate (2.7 liters), then trifluoroacetic anhydride (565 g) was added over 0.5 hour, keeping the temperature below 35°.
  • the iodonium trifluoroacetate salt can be used which is prepared as follows: Iodine (159 g) was suspended in trifluoroacetic anhydride (1 liter) and stirred under nitrogen whilst fuming nitric acid (350 ml) was added over 1.5 hours, keeping the temperature between 36° and 40°. Trifluoroacetic anhydride (300 ml) was then added and the mixture maintained at 40° under a stream of nitrogen until all nitrogen oxides were removed, then allowed to stand at room temperature overnight. The solvent was then removed under reduced pressure and the residual solvent removed by azeotroping with trifluoroacetic anhydride (2 X 300 ml).
  • the organic layer was dried over magnesium sulfate and concentrated to a residue consisting of the desired product, 3-[4-[4-(4- methylbenzoyl)benzoyl]phenyl]-l-propene, and tin-containing by-products.
  • This material was subjected to flash chromatography (silica gel, elution with 95:5 hexane : ethyl acetate) which removed most, but not all, of the tin impurities.
  • Aqueous sodium hydroxide (IN, 100 ml, 100 mmol) was added to a solution of 4-trans-aminomethyl-cyclohexyl-carboxylic acid (9.0g, 60 mmol), in dioxane (100 ml), water (100 ml) at 0 degrees C.
  • Boc anhydride (15.9 g, 66 mmol) was added and the reaction was warmed to rt and stirred overnight.
  • the solution was concentrated to 50 ml, then was diluted with EtOAc (100 ml) and acidified to pH 2 with adqueous KHSO. (IN).
  • the organic layer was then extracted with water (100 ml) two times, and the organics were concentrated in vacuo.
  • Kaiser oxime resin (20 g, 0.7 mmol/g loading, Advanced Chem Tech) was added to a solution of N-t-butyloxy carbonyl-trans-4-aminomethyl cyclohexyl carboxylic acid (5.0 g, 20 mmol) and DCC (4.4 g, 20 mmol) in methylene chloride (200 ml) and was gentle mixed at rt overnight. The solid was filtered and collected, then washed with methylene chloride (5 x 100 ml).
  • Trans-4-aminomethyl cyclohexyl (Kaiser oxime resin) carboxylate 200 mg was suspended in DMF (3.0 ml) and N-methyl morpholine (0.2 ml) and 4, 4'- benzophenone dicarboxylic acid (190 mg, 0.7 mmol) and HBTU (265 mg, 0.7 mmol) was added and the reaction was gently mixed for 3 h.
  • the solid was filtered and collected, then was washed with with DMF (3 x 20 ml), then water (3 x 20 ml), then was resuspended in DMF (3.0 ml) and N-methyl morpholine (0.1 ml) and 4,4'- benzophenone dicarboxylic acid (0.35 mmol) and HBTU (0.35 mmol) was added and the reaction was gently mixed for 3 h.
  • the solid was filtered and collected, then was washed with with DMF (3 x 20 ml), then water (3 x 20 ml), then methylene chloride (5 x 20 ml), then was dried under vacuum.
  • the inhibitory activity of compounds at the different human SH2 domains was determined in vitro using SH2 domains expressed as fusion proteins in E. coli.
  • the SH2 domains used herein were the human forms of the src SH2 domain, Grb2 SH2 domain, lck SH2 domain, fyn SH2 domain, SH-PTP2 SH2 domain, p85 SH2 domain and hep SH2 domain.
  • DETl (“defined epitope tag 1 ") (SEQ ID NO: 1) is an 11 amino acid sequence found in the Human Immunodeficiency Virus Type 1 (HIV-1) envelope protein gpl20 (or gpl60).
  • HIV-1 Human Immunodeficiency Virus Type 1 envelope protein gpl20 (or gpl60).
  • Monoclonal antibodies to various epitopes of HTV-1 gpl20 (or gpl60) are known in the art, see, for example U.S. Patent 5,166,050.
  • monoclonal antibody 178.1 (see, e.g., Thiriart et al., J. Immunol..142:1832-1836 (1989)), which was prepared by immunization of mice with a yeast-expressed HIV-1 gpl60 molecule from strain BH10 (Ratner et al., Nature.212:277-284 (1985)). This tag was used for detection of expression (by Western blot), for purification of the protein (by affinity chromatography), and for configuring assays in which the fusion protein was captured or immobilized using the 178.1 antibody.
  • DET2 is a hexa- histidine sequence tag (SEQ ID NO: 2) which binds to nickel-containing resins and was used for purification purposes. Spacer (SEQ ID NO: 3) was utilized to design a BamH 1 restriction site at the indicated position of the construct.
  • the term -ek- refers to a recognition sequence (SEQ ID NO: 4) for the enterokinase protease which provides for the optional removal of the tags from the SH2 domain, thus producing an SH2 domain that contains no extraneous amino acids.
  • SH2 domains which contain no extraneous amino acids are preferable to tagged protein for crystallography studies.
  • SH2 refers to the SH2 domains of different proteins.
  • each DETl-DET2-spacer-ek-SH2 was designed such that the indicated restriction sites (BamHl and Xbal) flank the spacer- ek-SH2 region, thereby allowing different spacer-ek-SH2 contructs to be readily substituted into any one of the vectors described in Procedures 2 or 3 below to create a DETl-DET2-spacer-ek-SH2 tagged protein.
  • the DNA sequence encoding each DETl-DET2-spacer-ek-SH2 constructs was also designed such that the entire tagged SH2 domain can be moved as an Ndel-Xbal fragment into any expression vector containing an Ndel site at an appropriate distance downstream of E.
  • E. coli expression vector pEAlKnRBS3 This vector is a derivative of the series of vectors described in Shatzman, A, Gross, M, and Rosenberg, M, 1990, "Expression using vectors with phage lambda regulatory sequences", In: Current Protocols in Molecular Biology (F.A. Ausubel et al , eds.), pp. 16.3.1-16.3.11, Greene Publishing and Wiley-Interscience, N.Y. (hereinafter F.A. Ausubel et al.). The specific vector pEAlKnRBS3 is described in Bergsma et al, 1991, J. Biol. Chem. 266:23204- 23214.
  • Procedure 1 Cloning and Expression of chicken src SH2 domain containing tags DETl and DET2 (DETl-DET2-spacer-SH2).
  • a DNA sequence encoding the tagged protein DETl-DET2-spacer-SH2 was PCR amplified from a cDNA clone containing the chicken src gene (p5H; Levy et al 1986. Proc. Natl. Acad. Sci. USA £2:4228) by methods well known to those skilled in the art by using the following primers:
  • the underlined sites are an Ndel recognition site (5') and a BamHl recognition site (3').
  • the underlined region is an Xbal recognition site.
  • the PCR product was digested with Ndel and Xbal, followed by isolation of the digested fragment on an agarose gel.
  • the fragment was ligated into Ndel-Xbal- digested pEAlKnRBS3 vector (Bergsma et al, supra) that had been agarose gel purified as a 6.5 kbp fragment.
  • the ligation reaction was used to transform __ ccji MM294cF (F.A. Ausubel et al., supra).
  • a plasmid containing an insertion of the correct fragment was identified and confirmed by DNA sequencing.
  • the resultant plasmid encodes DETl-DET2-spacer-SH2 under the control of the phage lamda P L promoter and regulatory system.
  • Plasmid DNA was purified from MM294cF and used to transform ⁇ _ so . strain AR120.
  • expression of the phage promoter can be induced by addition of nalidixic acid to the growing culture as described in F.A. Ausubel et al, supra. Nalidixic acid induction of AR120 containing this plasmid, followed by analysis of the cellular proteins on an SDS- polyacrylamide gel stained with Coomassie Blue (F.A.
  • Procedure 2 Cloning, expression and purification of human src SH2 domain containing tags and an enterokinase proteolytic cleavage site (DETl-DET2-spacer- ek-src SH2).
  • a DNA sequence encoding protein ek-src SH2 was PCR amplified from a cDNA clone containing the human src gene (c-src SH2 DNA sequence identical to that described in Takeya.T. and Hanafusa, H, 1983 Cell 32:881-890) using the following primers:
  • the underlined site is a BamHl recognition site.
  • the underlined region is an Xbal recognition site.
  • the PCR product was digested with BamHl and Xbal, followed by isolation of the digested fragment on an agarose gel. The fragment was ligated into BamHI- Xbal-digested expression vector containing the tagged chicken src gene DET1-
  • DET2-spacer-SH2 described in Procedure 1 above.
  • the BamHl site is located between the coding regions for DET2 and SH2, and the Xbal site is located after the 3' end of the SH2 coding region.
  • the ligation reaction was used to transform __ ___ MM294cI ⁇
  • the construct DETl-DET2-spacer-ek-src SH2 was confirmed by DNA sequencing (SEQ ID NO: 5) and induced in E. £ ⁇ _i strain AR120 as described in Procedure 1 above.
  • a Coomassie-Blue-stained, Western-blot- positive induced protein band with an apparent molecular weight of 16,000 was observed after nalidixic acid induction.
  • a DNA sequence encoding protein ek-lck SH2 was PCR amplified from a cDNA clone containing the human lck gene (Genbank accession number M36881) using the following primers:
  • the underlined site is a BamHl recognition site.
  • the underlined region is an Xbal recognition site.
  • the PCR product was digested with BamHl and Xbal, followed by isolation of the digested fragment on an agarose gel.
  • the fragment was ligated into BamHI- Xbal-digested expression vector containing the tagged chicken src gene DET1- DET2-spacer-SH2 described in Procedure 1 above.
  • the BamHl site is located in between the coding regions for DET2 and SH2, and the Xbal site is located after the 3' end of the SH2 coding region.
  • the ek-lck SH2 sequence replaced the src SH2 sequence in the above vector.
  • the ligation reaction was used to transform E. coli MM294cF.
  • Procedure 4 Cloning and expression of human hep SH2 domain containing tags and an enterokinase proteolytic cleavage site (DETl-DET2-spacer-ek-hcp SH2).
  • a DNA sequence encoding protein ek-hcp SH2 (hep SH2 DNA sequence identical to that described in Shen, S-H. Nature (1991) 5 : 736-739) was reverse transcriptase-PCR amplified from human fetal liver RNA.
  • RNA isolation used Tri- Reagent (Molecular Research Center Inc.) and the Reverse Transcriptase system (GEBCO-BRL) according to the manufacture's instructions. PCR was carried out using the following primers:
  • the underlined site is a Bglll recognition site.
  • the underlined region is an Xbal recognition site.
  • the PCR product was digested with Bglll and Xbal, followed by isolation of the digested fragment on an agarose gel.
  • the fragment was ligated into BamHI- Xbal-digested expression vector containing the tagged human src gene DET1- DET2-spacer-ek-src SH2 described in Procedure 2 above.
  • the BamHl site is located in between the coding regions for DET2 and ek
  • the Xbal site is located after the 3' end of the SH2 coding region.
  • the ek-hcp SH2 sequence replaced the ek-src SH2 sequence in the above vector.
  • the ligation reaction was used to transform E x £ ⁇ li MM294cF.
  • the construct containing DET 1 - DET2-spacer-ek-hcp SH2 was confirmed by DNA sequencing (SEQ ID NO: 7) and used to transform E. £Qli GI698 (Invitrogen Corporation, San Diego, CA).
  • Induction of the phage lambda promoter was induced by addition of tryptophan to the culture medium to 10 mg/ml, per the manufacture's instructions.
  • a Coomassie-Blue- stained, Western-blot-positive induced protein band with an apparent molecular weight of 15,000 was observed after tryptophan induction of cells growing at 30° C.
  • the SH2 domain purified in this fashion, was found to bind with high affinity in a specific, saturable fashion to the appropriate pY peptide in the "Binding Assays" described below, demonstrating that the tag did not interfere with function and that the protein was refolded successfully.
  • This expressed fusion protein, DETl-DET2-spacer-ek-hcp SH2 was utilized in the "Binding Assays” described below in order to determine the specificity of compounds to selectively inhibit the human hep SH2 domain.
  • Fusion proteins having the structure GST-X-SH2 were prepared as described in the GST gene fusion kit system available from Pharmacia (New Jersey).
  • GST is the tagging sequence glutathione s-transferase epitope (SEQ ID NO: 8) for fyn, Grb2 and SH-PTP2 and is the tagging sequence glutathione s-transferase epitope (SEQ ID NO: 9) for p85.
  • SH2 refers to the SH2 domains of fyn, Grb2, p85 and SH-PTP2 which were expressed and purified using glutathione Sepharose 4B (Pharmacia) according to "Current Protocols in Molecular Biology", ed. FM Ausubel et al., pub.
  • X is an appropriate linker, preferably of 6 to 21 base pairs, used to keep the SH2 construct in frame and complement cloning. As such, the sequence of X is not critical. One skilled in the art can readily construct the appropriate linker.
  • the DNA sequence encoding each GST-X-SH2 fusion protein was designed such that the indicated restriction sites (BamH 1 and EcoRI) flank the SH2 region.
  • the vector used in the instant experiments was the E. coli expression vector pGEX-2T (Pharmacia) for fyn, Grb2 and SH-PTP2, and pGEX-3X (Pharmacia) for p85. Each of these vectors result in SH2 constructs having additional C-terminal amino acids as described below.
  • the sequence encoding the SH2 domain of human fyn (amino acids 143- 252) (Yamamoto, T. et al. Proc. Natl. Acad. Sci. USA 83. 5459-5463 (1986)) was cloned into the BamHl and EcoRI sites of the expression vector pGEX-2T.
  • the SH2 domain including the additional C-terminal amino acids leucine-threonine- asparagine-serine-serine (SEQ ID NO: 10) was cloned by PCR techniques known to those skilled in the art to yield the expressed fusion protein GST-X-fyn. This expressed fusion protein was then utilized in the "Binding Assays" described below in order to determine the specificity of compounds to selectively inhibit the human fyn SH2 domain.
  • Human p85 SH2 domain The sequence encoding the SH2 domain of human p85 (amino acids 321-440) (Skolnik, E. et al., ___U __[, 83-90 (1991)) was cloned into the BamHl and EcoRI sites of the expression vector pGEX-3X.
  • the SH2 domain including the additional C-terminal amino acids asparagine-serine-serine (SEQ ID NO: 1 l) was cloned by PCR techniques known to those skilled in the art to yield the expressed fusion protein GST-X-p85. This expressed fusion protein was then utilized in the "Binding Assays" described below in order to determine the specificity of compounds to selectively inhibit the human p85 SH2 domain.
  • Human SH-PTP2 SH2 domain The sequence encoding the SH2 domain of human SH-PTP2 (amino acids l-106))(Bastien, L. et al., Biochem. Biophvs. Res. Commun. ____,, 124-133 (1993)) was cloned into the BamHl and EcoRI sites of the expression vector pGEX-2T.
  • Human Grb2 SH2 domain The sequence encoding the SH2 domain of human Grb2 (amino acids 58-159) (Lowenstein, E. et al., Cell-Z ⁇ , 431-442 (1992)) was cloned into the BamHl and EcoRI sites of the expression vector pGEX-2T.
  • the SH2 domain including the additional C-terminal amino acids isoleucine-histidine- arginine-aspartate (SEQ ID NO: 25) was cloned by PCR techniques known to those skilled in the art to yield the expressed fusion protein GST-X-Grb2.
  • a six nucleotide linker was used and resulted in the amino acids glycine and serine between the GST and SH2 domain. This expressed fusion protein was then utilized in the "Binding Assays" described below in order to determine the specificity of compounds to selectively inhibit the human Grb2 SH2 domain.
  • Binding Assays The potency of compounds at the SH2 domains was determined based on the ability of such compound to selectively inhibit such SH2 domain from binding to its respective specific pY peptide.
  • the binding assays for the SH2 domains and pY peptides were performed in an ELISA-based 96 well plate assay.
  • hydrophilic Durapore® pore size 0.65um Cat. No. MADVN6550
  • 2 ul 50% suspension
  • Protein-G Sepharose available from Pharmacia of N.J. Cat. No.
  • TBS-T 4°C
  • 90 ul of TBS-T were then added to each well.
  • Specific pY biotinylated peptides were diluted to a concentration of 1.0 uM in TBS- T (these peptides can be obtained from Bachem Bioscience of Pennsylvania, Genosys Biotechnologies of Texas and California Peptide Research of California). 10 ul was aliquoted per well to yield a final concentration of 0.1 uM (approx. the K ⁇ ⁇ for each SH2 domain/peptide pair) and a final volume of 100 ul.
  • the assay plates were incubated until equilibrium binding was attained (3 hr at 4°C with shaking).
  • the assay plates were washed 2 X per well TBS-T (4°C), then 100 ul of SABC (Strepavidin biotinylated horseradish peroxidase complex, available from the Zymed corporation of California cat. no. 93-0043, 1 drop reagent A (streptavidin) and 1 drop of reagent B (AH-biotin conjugated-horseradish peroxidase) per 10 ml of TBS- T, incubated at 37°C for 30 minutes, then cooled to 4°C) was added per well, then incubated at 4°C for 30-60 minutes. The plates were then washed 4 X with TBS-T (4°C) (250 ul/well)/wash).
  • SABC Stepavidin biotinylated horseradish peroxidase complex, available from the Zymed corporation of California cat. no. 93-0043
  • reagent A streptavidin
  • reagent B AH-
  • KI (IC 50 - Rtot+Rtot/2((*D/(KD+*D))+(KD/(KD+*D+Rtot/2)))/( 1 +*D/KD+Rtot/KD((KD+*D /2)/(KD+*D)))
  • KD is the dissociation constant for a ligand in a receptor ligand interaction, normally equaling the concentration of ligand which is at 1/2 Vmax on a saturation binding curve>
  • the pY peptide ligands used in the above Binding Assays are as follows:
  • Biotinylated pY peptide ligand containing an aminocaproic acid (Aca) linker used for p85 SH2 Asp-Gly-Gly-pTyr-Met-Asp-Met-Ser-Lys-Asp-Glu (SEQ ID NO: 14)
  • Biotinylated pY peptide ligand containing an aminocaproic acid (Aca) linker used for SH-PTP2 SH2
  • Tables I and II iUustrate the cross reactivity of SH2 antagonists at the indicated SH2 domains. From the results disclosed in these tables compounds which have binding affinities/inhibitory concentrations which are greater than fifty-fold higher at the lck SH2 domain than the binding affinities/inhibitory concentrations at other SH2 domains can be readily identified.
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • Lys Ser lie Arg lie Gin Arg Gly Pro Gly Arg
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE

Abstract

Cette invention présente une technique de traitement de maladies auto-immunes consistant à administrer à un patient une quantité thérapeutique utile d'un composé se liant à un domaine SH2 de lck humain avec une affinité de liaison de plus de cinquante fois plus élevée que l'affinité de liaison faisant se lier ce composé à un domaine SH2 du src humain et à un domaine SH2 de fyn humain. Ce composé se lie également à un domaine SH2 de hcp humain, à un domaine SH2 du Gbr2 humain, à un domaine SH2 de SH-PTP2 humain ainsi qu'à un domaine SH2 de p85 humain avec une affinité de liaison de plus de cinquante fois plus faible que l'affinité de liaison faisant se lier ce composé à ce domaine SH2 de lck humain.
PCT/US1996/001964 1995-02-10 1996-02-09 Utilisation de composes specifiques comportant lck sh2 pour le traitement de maladies auto-immunes et du rejet d'allogreffes WO1996024343A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
EP96905494A EP0809490A4 (fr) 1995-02-10 1996-02-09 Utilisation de composes specifiques comportant lck sh2 pour le traitement de maladies auto-immunes et du rejet d'allogreffes
BR9607614A BR9607614A (pt) 1995-02-10 1996-02-09 Uso de compostos específicos para sh2 de lck para tratar doenças autoimunes e rejeição ao aloenxerto
AU49237/96A AU4923796A (en) 1995-02-10 1996-02-09 Use of lck sh2 specific compounds to treat autoimmune diseases and allograft rejection
JP8524486A JPH10513474A (ja) 1995-02-10 1996-02-09 自己免疫疾患および同種異系移植拒絶を治療するためのｌｃｋ ｓｈ２特異的化合物の使用
FI973259A FI973259A (fi) 1995-02-10 1997-08-07 LCK-SH2-spesifisten yhdisteiden käyttö hoidettaessa autoimmuunisairauksia ja kudossiirrännäisten hyljintää
NO973659A NO973659L (no) 1995-02-10 1997-08-08 Anvendelse av LCK SH2-spesifikke forbindelser til behandling av autoimmune sykdommer og transplantatavvisning
MXPA/A/1997/006130A MXPA97006130A (en) 1995-02-10 1997-08-11 Use of specific compounds of lck sh2 to treat autoimmune diseases and the rejection of aloinjer

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US38638195A 1995-02-10 1995-02-10
US08/386,381 1995-02-10
US40022095A 1995-03-07 1995-03-07
US08/400,220 1995-03-07
US49735795A 1995-06-30 1995-06-30
US08/497,357 1995-06-30

Publications (1)

Publication Number Publication Date
WO1996024343A1 true WO1996024343A1 (fr) 1996-08-15

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PCT/US1996/001964 WO1996024343A1 (fr) 1995-02-10 1996-02-09 Utilisation de composes specifiques comportant lck sh2 pour le traitement de maladies auto-immunes et du rejet d'allogreffes

Country Status (10)

Country Link
EP (2) EP0809490A4 (fr)
JP (2) JPH10513474A (fr)
KR (1) KR19980702115A (fr)
AU (1) AU4923796A (fr)
BR (1) BR9607614A (fr)
CA (1) CA2212645A1 (fr)
FI (1) FI973259A (fr)
HU (1) HUP9802078A3 (fr)
NO (1) NO973659L (fr)
WO (1) WO1996024343A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5922697A (en) * 1996-10-02 1999-07-13 Warner-Lambert Company Compounds, compositions and methods for inhibiting the binding of proteins containing an SH2 domain to cognate phosphorylated proteins
WO2004085406A1 (fr) 2003-03-24 2004-10-07 F. Hoffmann-La Roche Ag Benzyl-pyridazinones en tant qu'inhibiteurs de transcriptase inverse
US7288542B2 (en) 2004-03-23 2007-10-30 Roche Palo Alto Llc Non-nucleoside reverse transcriptase inhibitors

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU5136093A (en) * 1992-09-25 1994-04-26 Warner-Lambert Company Peptide antagonists of sh2 binding and therapeutic uses thereof
US5580979A (en) * 1994-03-15 1996-12-03 Trustees Of Tufts University Phosphotyrosine peptidomimetics for inhibiting SH2 domain interactions
US5710129A (en) * 1995-02-23 1998-01-20 Ariad Pharmaceuticals, Inc. Inhibitors of SH2-mediated processes

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF CELL SCIENCE, 1994, Vol. 18, MARENGERE et al., "Structure and Function of SH2 Domains", pages 97-104. *
JOURNAL OF MEDICINAL CHEMISTRY, 1995, Volume 38, BURKE Jr. et al., "Conformationally Constrained Phosphotyrosyl Mimetics Designed as Monomeric Src Homology 2 Domain Inhibitors", pages 1386-1396. *
See also references of EP0809490A4 *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, 13 January 1995, Vol. 270, No. 2, WANGE et al., "F2(Pmp)2-TAMXi3, a Novel Competitive Inhibitor of the Binding of ZAP-70 to the T Cell Antigen Receptor, Blocks Early T Cell Signaling", pages 944-948. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5922697A (en) * 1996-10-02 1999-07-13 Warner-Lambert Company Compounds, compositions and methods for inhibiting the binding of proteins containing an SH2 domain to cognate phosphorylated proteins
WO2004085406A1 (fr) 2003-03-24 2004-10-07 F. Hoffmann-La Roche Ag Benzyl-pyridazinones en tant qu'inhibiteurs de transcriptase inverse
US7189718B2 (en) 2003-03-24 2007-03-13 Roche Palo Alto Llc Non-nucleoside reverse transcriptase inhibitors
US7288542B2 (en) 2004-03-23 2007-10-30 Roche Palo Alto Llc Non-nucleoside reverse transcriptase inhibitors

Also Published As

Publication number Publication date
EP0809490A4 (fr) 1999-10-20
NO973659D0 (no) 1997-08-08
CA2212645A1 (fr) 1996-08-15
EP0809490A1 (fr) 1997-12-03
HUP9802078A3 (en) 1999-04-28
EP0811159A1 (fr) 1997-12-10
FI973259A (fi) 1997-10-08
JPH10513474A (ja) 1998-12-22
KR19980702115A (ko) 1998-07-15
BR9607614A (pt) 1998-06-09
JPH10513564A (ja) 1998-12-22
AU4923796A (en) 1996-08-27
FI973259A0 (fi) 1997-08-07
NO973659L (no) 1997-10-08
HUP9802078A2 (hu) 1998-12-28

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